Diethyl allyl phosphonate as an anti-microbial agent

ABSTRACT

Diethyl alpha allyl phosphonate can be used to inhibit and/or prevent growth of undesirable algae, bacteria, fungi, yeast, and other microorganisms. This invention is particularly concerned with the bacteriostatic and bactericidal properties of diethyl alpha allyl phosphonate compounds against species of Proteus, Salmonella, Shigella, Staphylococcus and Xanthomonas.

United States Patent [191 Kerst et al.

[ Jan. 1,1974

[ DIETHYL ALLYL PHOSPHONATE AS AN ANTI-MICROBIAL AGENT [75] Inventors: Al F. Kerst; John D. Douros, Jr.,

both of Littleton, Colo.

[73] Assignee: The Gates Rubber Company,

Denver, C010.

[22] Filed: Oct. 28, 1970 [21] Appl. No.: 84,903

[52] US. Cl. 424/219, 71/67, 71/86 [51] Int. Cl. A01n 9/36 [58] Field of Search 424/219 [56] References Cited UNITED STATES PATENTS 4/1967 Fitch et a] 424/222 X OTHER PUBLICATIONS Hackhs Chemical Dictionary, pg. 29 (1969).

Ford-Moore & Williams J. Chem. Soc, pp. 1465-1467 (1947).

Primary Examiner-Albert T. Meyers Assistant Examiner-Leonard Schenkman Att0rney-Raymond Fink, H. W. Oberg, Jr. and Curtis l-l. Castleman, Jr.

[5 7] ABSTRACT 52 Claims, No Drawings DIETHYL ALLYL PHOSPHONATE AS AN ANTl-MICROBIAL AGENT BACKGROUND OF THE INVENTION While there is no dearth of organophosphorous microbial growth inhibitors existing today, the antimicrobial properties of diethyl alpha allyl phosphonate have not been previously discovered. Furthermore, few of the commercially available organophosphorous growth inhibitors offer diethyl alpha allyl phosphonates activity against such a broad spectrum of algae, bacteria, fungi, yeast, et cetera. This broad activity is often desirable since the inhibition of one microorganism species or group of species may create an imbalance which often results in the rampant growth of other deleterious microorganisms.

SUMMARY OF THE INVENTION According to the present invention, it has been found that diethyl alpha allyl phosphonate PCH CH=CH ll C2H -.O O

is a very effective antimicrobial agent. The compound diethyl alpha allyl phosphonate is well known and may be prepared according to the methods disclosed by Ford-Moore, A. H. and Williams, J. H., J. Chem. Soc. 1947, 1465. The diethyl alpha allyl phosphonate used to establish the present invention was prepared by two different procedures. The first procedure involved the reaction of substantially equimolar proportions of sodium diethyl phosphite and allyl bromide in ethanol. The reaction took; place smoothly in the temperature range of from to 100 C., with the preferred reaction conditions being from 20 to 65 C. for about 1 hour. Following completion of the reaction, the precipitated sodium bromide was removed by filtration and the ethanol was evaporated off under vacuum. The remaining syrup was distilled under vacuum and produced a 65 to 70 percent yield of diethyl alpha allyl phosphonate.

In an alternative procedure, the diethyl alpha allyl phosphonate compounds of the present invention were prepared by reacting triethyl phosphite with allyl bromide according to the Arbusov method. Good results were obtained by employing substantially equimolar proportions of the reactants. This reaction took place readily at temperatures of from 90 to 100 C. to produce the diethyl alpha allyl phosphonate and a ethyl bromide by-product which was collected by a dry ice condenser. The final mixture was distilled to give an 85 to 90 percent yield of the diethyl alpha allyl phosphonate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the antimicrobial qualities of diethyl alpha allyl phosphonate and describe how these antimicrobial qualities may be utilized in various phases of agriculture, animal husbandry, pharmacology and water treatment technology.

EXAMPLE 1 Antibacterial and Antiyeast Activity The in vitro effectiveness of diethyl alpha allyl phosphonate against bacteria and yeast species was established in the following manner. One loopful of each of the investigated bacteria or yeast was transferred from agar slants to 10 ml. of trypticase soy broth and incubated at 37 for 18 hours. At the end of this period, the bacteria or yeast was seeded into the same medium (1.52 percent agar) in which the original inoculum was prepared. The bacteria were then seeded at 1 ml. of inoculum per 250 ml. of medium, which were equivalent to at least 1 X 10 cells/ml. determined by dilution platecount or nephelometer readings. The resultant mixtures were poured into heat-resistant sterile petri dishes at a temperature of 45 C. Analytical filter paper discs 1.2 cm. in diameter were used for the agar diffusion technique. Each disc was saturated with 0.08 ml. of the solubilized diethyl alpha allyl phosphonate compound ug./disc) and placed on the surface of the hardened agar. The plates were incubated at 37 C. for 18 hours. The activity of the diethyl alpha allyl phosphonate compounds was established by measuring the zone of inhibition in centimeters. Untreated control plates were used as a basis for comparison and these exhibited a profuse growth of bacteria. The results of these tests were as follows:

Gram positive and Gram negative bacteria Zone of Inhibition in centimeters Streptococcus hemolytic 2.3 Group A Streptococcus hemolytic 2.4 Group B X amhomonas phaseoli 5 .3 ATCC No. 9563 Staphylococcus aureus 1 .6 ATCC No. 2091 Escherichia coli 2.8 ATCC No. 9637 Shigella boydii 4.8 ATCC No. 9212 Shigella baydii 4.6 ATCC No. 9905 Shigella sonnei 3.8 MMV 6654 Shigella flexneri TYPE 6 4.9 MMV 760 Shigella flexneri TYPE 4 3.8 MMV 6625 Shigella dysenleriae TYPE 2 3.9 MMV 6673 Salmonella panama 4.8 ATCC No. 7378 Salmonella paratyphi 4.7 ATCC No. 9281 Salmonella enteritis 4.0 ATCC NO. 13076 Salmonella pullarum 3.7 ATCC NO. 10398 Salmonella derby 3.6 ATCC No. 6960 Salmonella gallinarium 3.0 ATCC No. 9184 Salmonella ryphimurium 4.8 SR-l 1 Salmonella lyphosa 4.9 ATCC No. 19403 Neisseria gonorrhoeae 5.1 ATCC No. 19424 Neirseria inlraeellularis 6.1 ATCC No. Available on Request Neisseria meningilides 3.3 ATCC No. 13077 Listeria monucymgenes 1.5 ATCC No. 15813 Vibrio fetus 3.7 ATCC No. 15296 Vibrio cholerae 3.5

ATCC No. 14035 Proteus vulgaris 3.3 ATCC No. 4984 Erwim'a caromvora 3.6 ATCC No. 495

Myrubacterium butyricum 2.3 ATCC No.11314 Mycvbacterium fartuitum, Debos 2.4 ATCC No. 4243 Myculmcterium avt'um 1.9 ATCC No. 19421 Mycabacterium bovt's 2.0 ATCC No. 19274 Mycohactertum phlei 2.8 ATCC No. 11782, phage host Klehsiella pneumoniae 2.7 ATCC No. Available on Request Micmcoccus tetragena 2.9 ATCC No. 10875 Micrococcus melitensis 3.5 ATCC No. 19399 Micmcoccus lysodeiktl'cus 3.7 ATCC No. 4698 Corynebacterium diphtheriae 2.2 ATCC No. 19409 Corynebactert'urn haemolyticum 2.2 ATCC No. 9345 Diplococcus intracellularis 2.7 ATCC No. Available on Request Diplococcus pneumoniae 2.5 ATCC No. 6303 Hemophilus hemolyticus 3.6 ATCC No. 10014 Hemuphilus influenzae 3.6 ATCC No. 19418 Hemopht'lus parainfluenzae 3.6 ATCC No. 7901 Hemopht'lus sul's 3.5 ATCC No 19417 Hemophilus vagtnalt's 3.6 ATCC No. 14018 Bacternides nigrescens 0.9 ATCC No. Available on Request Baetemides pneumosintes 1.0 ATCC No. Available on Request Bacterot'des serpens 1.0 ATCC No. Available on Request Brucella abortus 1.0 ATCC No. 4315 Brucelln melt'tensis 1.0 ATCC No. 19396 Brucella suis 1.0 ATCC No. 4312 EXAMPLE 11 Secondary Screen Antibacterial Activity To further define the scope of this invention, secondary screening tests using the techniques described in the primary screening tests were also employed against selected bacteria genera at various other concentrations. The results of these secondary screens were as follows:

Compound: Diethyl alpha allyl ATCC No. 9905 Shigella flexneri Type 6 4.4 3.0 2.0h MMU 760 Shigella flexneri Type 4 3.3 2.7 2.2 MMU 6625 Shigella dysenteriae Type 2 3.7 2.7 '2.2 MMU 6673 Shl'gella sonnet- 3.6 2.6 1.8 MMU 6654 Pseudomonas aeruginosa l 9 0 0 ATCC No. 10145 Pseudomonas aerugtnosa 2.4 0 0 ATCC No. 8709 Pseudomonar uerugirtosa 3.5 2.2h 0 ATCC NO. 12055 Pseudomonas aerugt'nosn 3.6 2.9h 1.8 28

Pseudomonas multnphilia 4.3 2.7 1.7 CI 107 Pseudomonas K997 2.9 2.6h h Pseudomonas K966 3.0 2.8 h

Proteus vulgaris- 2.8 2.0 0 ATCC No. 9484 Proteus vulgaris 3.4 3.0 2.3 ATCC No. 11427 Proteus vulgart's 2.7 2.2 1.6 ATCC N0. 4699 Proteus vulgaris 3.1 2.7 1.7 ATCC No. 6896 Proteus vulgaris 2.7/3.3 2.6 1.8 ATCC No. 9920 Proteus vulgaris 2.7 2.1 0 ATCC No. 6897 Proteus vulgaris 3.4 2.6 1.7 ATCC No. 12454 Proteus mirabilis 1.6 1.3 tr ATCC No. 9961 Proteus murganii G951 2.2 1.8h

Proteus mirabilo- G912 3.0 2.2h tr h Proteus mirabilis K723 2.8 2.3h tr h Comments: h hazy zone This data indicates that diethyl alpha allyl phosphonate compounds can be used to inhibit many important types of diseases. For example, they can be used against:

Bacteria Staphylococcus aureus Streptococcus faecalts Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Erwl'nia cartovora Xanthomonas phaseoli The demonstrated antibacterial activity of diethyl alpha allyl phosphonate compounds against Staphylococcus aureus and Escherichia coli is of particular interest to the field of pharmacology since the disclosed activity indicates that these compounds can be formulated as powders, salves, and ointments for administration in the treatment of burns and bacterially induced inflammations such as abscesses, dermatitis, rashes and the like, particularly in domestic animals.

Although the precise mode of action whereby diethyl allyl phosphonate inhibits bacteria growth is not completely understood, it is believed that the diethyl alpha allyl phosphonate compounds of this invention may serve as chemical antagonists; that is, as chemicals which compete with enzymes essential to the development of such bacteria. Since enzymes perform their catalytic function by virtue of their affinity for their natural substrate; any compound resembling a substrate in its chemically critical aspect may also have an affinity for the enzymes. If this affinity is great enough, the analog will displace the normal substrate from the enzyme-and will prevent the growth reaction from taking place. It is believed that diethyl alpha allyl phosphonate has a chemical affinity for an essential site on one enzyme necessary for bacterial growth and life.

The diethyl alpha allyl phosphonate formulations of this invention can also contain other therapeutically valued supplements such as local anesthetics, irradiated oils, and other medicinal substances. When used for these or similar purposes, diethyl alpha allyl phosphonate may be incorporated in any therapeutically acceptable carrier such as oils, salves and ointments, together with adjuvants comprising surface active agents, detergents, dispersing agents, stabilizers and other modifiers which may facilitate the handling and application of the antibacterial material. In the case of the in vitro applications of the compositions of this invention, it is difficult to predict with precision what in all cases will constitute a therapeutic dose even on a weight basis. Variable factors such as type, duration and severity of infection and mode of administration may be determining factors for the establishment of therapeutic doses.

Those skilled in the art will recognize that the above data indicates that the scope of this invention should not be limited to any particular disease species or to any particular type of animal or plant life. For example, the noted activity of diethyl alpha allyl phosphonate against Xanthomonas phaseoli suggests that this compound will also prove to be of value against such other Xanthomonas species as Xanthomonas transluscens, Xanthomonas juglandis, Xanthomonas vesicatoria, Xanlhomonas barbareae, Xanthomonas pelargonii, Xanthomonas alfalfae, Xanthomonas vasculorum, et cetera. Xanthomonas species are known to cause diseases of tomatoes, sugar cane, rice, sugar beets, cotton, walnuts, wheat, rye, barley, and beans. Some of the more noteworthy Xanthomonas related diseases are X anthomonas vesicatoria (Bacterial Leaf Spot of Tomatoes), Xanthomonas phaseoli (Common Bacterial Blight of Bean), Xanthomonas vasculorum (Gumming Disease of Sugar Cane) and Xanthomonas mal vacearum (Bacterial Blight of Cotton).

EXAMPLE III Inhibition of Xanthomonas Vesicatoria The efficacy of diethyl alpha allyl phosphonate under simulated field conditions against a bacteria such as Xanthomonas vesicataria (Bacterial Leaf Spot of Tomato) was established in the following manner. A diethyl alpha allyl phosphonate composition was prepared for spraying by dissolving it in a suitable solvent such as acetone, methyl alcohol or ethyl alcohol and then diluting the solution to the desired concentration with deionized water containing wetting and dispersing agents. Tomato seedlings, Bonny Best variety, in 7-leaf to 8leaf growth stage, were mounted on a compound turntable and sprayed at 35 .pounds pressure for 50 seconds with diethyl alpha allyl phosphonate solutions at the concentrations indicated below. After drying, the

treated plants were spray inoculated at 30 pounds pressure with an aqueous cell suspension of Xanthomonas vesicatoria containing five per cent Carborundum and then immediately placed in an incubation chamber maintained at F. and percent plus relative humidity. After 40 hours in the incubation chamber, the plants were removed to the greenhouse for further development of the infection. Disease severity was determined by counting the lesions present on the treated leaves. The effectiveness of treatment was determined by direct comparison of the treated plants with the untreated ones. Streptomycin sulfate was used as a reference control antibacterial agent. The results of these tests were as follows:

% Inhibition at 500 ppm: 54% Inhibition at 1000 ppm: 54% Control Streptomycin Control Concentration: I00 ppm EXAMPLE IV Antifungal Activity The antifungal activity of diethyl alpha allyl phosphonate compounds was established by treating F usarium oxysporum, F usarium roseum, Rhizopus nigricans, Rhizopus stolonifer, Aspergillus niger and Alternaria solani test fungi in the following manner:

One loopful of each of the tested viable fungi cultures, spores and mycelia was transferred from an agar slant to an 80 ml. portion of the nutrient broth composed of oatmeal agar, Czapeks, Sabouraud and deoinized water to volume. The 80 ml. portion of the fungi and broth was then placed in a sterile shake flask (300 ml.) and the flask was placed on a rotary shaker for 96 to hours at room temperature. At the end of this incubation time period, 10 ml. of the liquid were homogenized and placed into another sterile shake flask (300 ml.) containing 80 ml. of the above nutrient broth and 60 ppm of the inhibitor being evaluated. The flasks were placed on a rotary shaker operating at 240 rpm at room temperature for 3 to 9 days. After this second incubation time, the flasks are taken off and examined for visible fungal growth and mycelial weights are determined. Untreated controls were used as the basis of comparison and these displayed profuse fungal growths containing species of F usarium, Rhizopus, Aspergillus and Alternaria. The results of these tests indicated that the diethyl alpha allyl phosphonate compounds of this invention imparted a substantial degree of inhibition of fungal growth at 60 ppm.

EXAMPLE V Antifungal and Antiyeast Activity To further define the antifungal activity of diethyl alpha allyl phosphonate the seeded agar plates were prepared by transferring the cultures from slants washed with saline or phosphate buffer and then transferred to the surface of hardened Sabouraud-Dextrose agar plates. Again, as in the case of Example I, the diethyl alpha allyl phosphonate was tested by impregnating filter paper discs (1.27 cm. in diameter) with 0.08 ml. of the solubilized diethyl alpha allyl phosphonate compounds I00 ug/disc) and placed on the surface of the hardened agar. The plates were incubated at 30 C. for 18 hours. The activity of diethyl alpha allyl phosphonate was established by measuring the zone of inhibition in centimeters. Untreated control plates were used as a basic for comparison and these exhibited a profuse growth of bacteria. The results of these tests U were as followsl Cladosporium trichoides 6.0 3.0 0

Zone of lnhibilion Microorganism in Ccntimetcrs Tm'ula bcrgc" L8 0 Aspergillus niger 2.4 cu ATCC NO. 1004 Ahemaria l i 2'4 Monosporum apmspcrmum 4.1 2.9 0 ATCC N0. 6396 0U Rhizupus smlom'fer 2.9 ATCC No. 10404 Allernan'a solani 3.3 1.8 0 Fusarium oxysporum 3.0 ATCC 639 UFCC 1122 10 Candida albicans 3.0 Gwl'whum P- 0 SR1 523 OU Verticillium albo arrum 3.8 2.3 1.0 ATCC No. 10833 n Seco ddry Fungal Screen Trichophyton mentagrophytes 4.8 3.5 2.1

ATCC No. 9129 A secondary screen usmg the techniques of Example Trichophywn mentagwphyms 50 349 V produced the following results at the dlethyl alpha ATCC 8215 allyl phosphonate concentrations indicated: Trichophyton wflflurans ATCC No. 10217 C(mccmrallon lg/disc 20 Cercospora betieola 3.5 2.0 2.6 MICROORGANISM 85.0 28.0 8.0 ATCC No 12825 Rhodotnrula sp. 2.0 1.6 0 Duke Pythium arrhenomanes 3.1 2.1 0

. ATCC No. 12531 Rhlzopur smlonlfer 3.0 1.8 0 ATCC 0404 Helminthosporium oryzae 2.7 2.4 0

ATCC No. 11000 Mumzscus purpurea 4.5 2.2 0 CU Comments: ng no growth.

slrcaguxmlsrzseum 3.8 1.2 0 Fusarium oxysporum cubense 4.0 3.0 0 S d C S UFCC H22 econ ary an 1 a creen scopulariopsis 3 4 2 2 u A secondary screen of yeast of the genus Candida 0U produced the following results when the techniques of Aspflgmus is 2 8 l 6 0 Examples V and VI were employed at the concentra- SR1 tions indicated: Aspergillus niger 3.3 tr 0 gf e'g' i g g y' Concentration (ug/disc) N Aspergillus nidulans 5.8 tr 0 MICROORGA [SM 53 0 l7 0 ATCC N0 0074 Candzda allncans 2.5 1.7h

g gg 221: o Candida albicans 2.3 1.7

. ATCC No. 752

g g zt gs 0 40 Candida 1111mm 3.0 1.711

. ATCC No. 11651 Aspergrllu: fumlgmus 3.2 2.0 0 Candida albicans 23 Lgh Duke 2548 g 0 Candida dlbiCllnA 2.5 1.911

Mt. Vernon SR1 Z20 Phnma pigmenmvora 4.5 3.0 2.0 a i f 22 Candida sp. 2.2 1.7 Pazcilomyces variuu' 5.1 4.0 0 2 :g 788 2 2 1 7 ATCC No. 1114 Mt. Vernon 512 Nigrm-para sphaerica 3.1 2.5 0 f qy 976 25h ATCC No. 11687 Candida sp. 2.2 1.7 Mt. Vernon 950 Absidm .rpinnra 3.7 2.1 0

Candida kruxer 1.0 0 ATCC No. 6648 0U JV Mucor racemosus 4.0 2.7 0 gum, Candida rmpicalis 2.2 1.6

. Duke 2847 22 2 2522 clcgans tr Candida psuedntmpicali: 1.9 tr

Duke 2543, U. Columbia Phycomyces nitens 3.5 1.9 tr g gb g A'ICC No. 9984 Candida zeylanoldes 3.5 2.6 Duke 2609 0 Comments: h hazy zone Penicillium notatum 6.4 3.5 tr H DU 8 b 5 0 2 3 o It will also be recognized by those skilled in the art $f,','f that other protectant, systemic and eradicant proce- Beam/aria tenclla 5.1 3.0 2.6 dures may provide detection of other biological activi- Mv ties. Patho ens re resentative of Ph com cetes, Asco- P y Y Phialophora verrucosa 5.2 2.0 tr mycetes, Basidiomycetes and the Fungi Imperfecti may provide indices of other fungicidal activity. Additional pathogens and appropriate host plants may well afford other opportunities to further define the degree and spectrum of the activity disclosed in this invention. Since no firm rules of procedure can be laid down for the sequence of such evaluations or for the choice of pathogens, diethyl alpha allyl phosphonate must be considered on the basis of its demonstrated performance in such primary evaluations and then progressively judged in subsequent studies. A wide range of pathogens, representative of economically important diseases, can be used to help define diethyl alpha allyl phosphonates biological activity and to assure high degress of success under field conditions. The following disease organisms, crops and reference standards may be used in such evaluations:

Disease Disease Organism Reference Compound Powdery Mildew of Erysiphe cichoracearum Maneb, Cucumbers Karathane Leaf Rust of Wheat Puccinia rubigo-vera Maneb, Karathane Leaf Rust of Wheat Puccinia rubigo-vera Plantvax Rice Blast Disease Piricularia oryzae Blasticidin Downy Mildew of Sugar Beet Peronosporn .rchnctii Karathane Downy Mildew of Lima Bean Phytophrhora phnseali .Karathane Bean Rust Uromyces phasenli var. Maneb typica Powdery Mildew of Wheat Erysl'phe gramim's Karathane Powdery Mildew of Apple Podosphaera leucmricha Karathane Powdery Mildew of Roses Sphaemthecu pannosa var. Karathane rosae Powdery Mildew of Erysiphe cicoracearum Karathane Cantalope Leaf Spot of Wheat Helmimhosporium szm'vum Maneb Early Blight of Tomato Allernan'a solani Maneb Rice Blast Disease Piri'cularia uryzae Blasticidin Cerocspora Leaf Spot of Sugar Beets Cercospora beticala Maneb Septoria Leaf Spot of Celery Septori'a apii-gmveolentis Maneb Apple Scab Venluria inaequall's Cyprex Common Bacterial Blight of Bean Xanthomonas phaseoli' Streptomycin Sulfate Wherever possible, the applicants recommend the use of in vivo procedures to test the diethyl alpha allyl phosphonate compositions of this invention todemonstrate their efficacy under more realistic conditions. However, not all pathogens lend themselves to such techniques. In order to provide additional spectrum definitions, the following fruit-rotting, storage decay and bacterial pathogens may be tested by in vitro" methods:

Brown Rot of Stone Fruits Sclemtinia fructicola Captan Grey Mold of Fruit and Vegetables Botryis cinerea Maneb Rhizopu'; Fruit and Vegetable Rot Rhizopus nigricans Maneb Citrus Green Mold Penicillium digimtum Maneb Citrus Blue Mold Penicillium italicum Karathane Bacterial Disease of Many Fruit crops Fseudamonas syringae Captan Bacterial Soft Rot Erwinia caralamra Captan In their plant protection aspects, the diethyl alpha allyl phosphonate compounds of this invention may be used in the manner known to the organophosphorus crop protection art; that is, they can be made up in solid or liquid formulations. Examples of solid formulations are dust, wettable powders, granules and pellets. As a dust, diethyl alpha allyl phosphonate compounds may be dispersed in powdered solid carriers such as ta'lc, soaps, soapstone, attapulgus clay as well as other finely divided solids known to the dusting art. When formulated as wettable powders, the active component may be employed in conjunction with inert fillers which may be of the clay type carrier or non-clay type, in conjunction with various combinations of wetting agents and emulsifiers which permit the adaptation of the concentration as a free-flowing powder for dispersion in the field.

Each of these carriers may in turn contain other carriers or extenders which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like as well as diatomaceous earth, micas or other suitable materials. When liquid formulations are desired, liquid extenders, dilutants or carriers of a non-reactive nature may be utilized. Examples of such materials are alcohols, ketones, glycols, aromatic hydrocarbons, petroleum fractions such as octane and various other distillates. From these considerations, it will also be recognized that the above formulations with slight modifications may be used in the field of animal husbandry as dusting powders and salves.

Where it is desired to use the aforementioned wettable powders or liquid formulations, either emulsified, dispersed or suspended in water or other fluids, one or more of the class of materials herein referred to as adjuvants can also be incorporated into the powder, dust or liquid formulation. These adjuvants comprise surface active agents, detergents, wettable agents, stabilizers, dispersing agents, suspending agents, emulsifying agents, spreaders, stickers and conditioning agents generally. To their modifying characteristics these adjuvants may facilitate handling and application and infrequently enhance or potentiate the diethyl alpha allyl phosphonate compositions of this invention in their biological activities by mechanisms which are frequently not well understood. A satisfactory but not exhaustive list of these adjuvants appear in Soap Chemical Specialties, Volume 31, No. 7, Page 61; No. 8, Pages 38-61; No. 9, Pages 52-67; and No. 10, Pages 38-67 (1955). See also, Bulletin No. 607 of the Bureau of Entomology and Plant Quarantine of the United States Department of Agriculture.

An additional advantage of diethyl allyl phosphonate is its compatibility with a variety of other biocidal and fungicidal materials. For example, it may be convenient to combine diethyl alpha allyl phosphonate with one or more other adjuvants, carriers, pesticides, biocides or fungicides of various structures. For example, diethyl alpha allyl phosphonate fungicidal inhibitors may be combined with insecticidal materials such as chlordane, benzene hexachlorides, DDT, DDD, the insecticidal carbamates, polychlorinated terpenes, parathions, methozychlor, insecticidal phosphates, phosphorothioates, phosphorodithioates and with fungicides such as sulphur, quinones, dodecylgaunidine and metal dimethyldithio-carbamates. 7

There are many other considerations such as concentration and method of application which may make some methods of application more favored than others. These considerations may include the type of organisms on which the compound is to be administered, the degree of activity, the degree of activity toward the particular organism, and side effects. Also to be considered is the cost of production and the characteristic solubility of diethyl allyl phosphonate in the carrier material.

The broad spectrum of antifungal activity afforded by diethyl alpha allyl phosphonate can also be utilized in the formulation of disinfectant solutions, paints, coatings, films and polymeric materials in order to protect against disease and rot caused by various fungi species. When used as a disinfectant, suitable formulations may be prepared by mixing the compound with an emulsifying agent in the presence of organic solvents and then diluting it with water to form an aqueous emulsion containing the diethyl alpha allyl phosphonate. Suitable emulsifying agents include, e.g., alkylbenzenesulfonates, polyalkene glycols, et cetera. Aqueous emulsions of diethyl alpha allyl phosphonate are particularly suited for use in disinfectant solutions used in washing hospital floors and walls. The following examples will further illustrate the antifungal properties of diethyl allyl phosphonate.

EXAMPLE VIII Preparation of a Vinyl Coating Resistant to Mildew Deterioration A vinyl coating is prepared using a commercially available preparation without a fungal growth inhibitor.

An identical vinyl coating was prepared except that 2 percent by weight of diethyl alpha allyl phosphonate was incorporated into the coating formulation.

Two sets of components such as asbestos tubing, silkwrapped transformers and rayon-wrapped solenoids were obtained. One set was sprayed with the vinyl coating containing inhibitor, the other with the identical coating without inhibitor.

EXAMPLE IX Preparation of Plasticizers Resistant to Mildew A commercial thermoplastic monomer was divided into portions which were treated as follows.

Portion i: To the first portion was added 2 percent by weight of diethyl alpha allyl phosphonate and percent by weight of dimentylnaphthalate as plastieizer. The monomer is polymerized and molded into 3-inch diameter discs, one-fourth inch in thickness prior to testing.

Portion 2: To this portion was added 2 percent by weight of diethyl alpha allyl phosphonate and 10 percent by weight of butyl isodecylphthalate as plasticizer. The monomer is polymerized and molded as above.

Portion 3: This portion was the untreated control of Portion 1 containing no fungal inhibitor but 10 percent by weight of dimethylphthalate as plasticizer. Again, the polymerization and molding are identical.

Portion 4: This portion was the untreated control of Portion 2 containing no fungal inhibitor but l0 percent by weight of butyl isodecylphthalate as plasticizer. The polymerization and molding are as described above.

The two plasticizers were chosen on the basis of their known susceptibility to Fusarium attack under high humidity and temperature conditions.

EXAMPLE X Vinyl Coatings and Plasticizers For Fungal Resistance The vinyl coated articles and controls of Examples VIII and IX were placed in an air-tighthigh temperature and high humidity chamber maintained at 80 F. and 95 percent humidity to simulate tropical temperature and humidity conditions. After a months exposure the vinyl coated articles treated with inhibitor were only slightly attacked by rot while the articles coated with vinyl without inhibitor were well rotted. The two untreated control polymer discs are examined and were found to be blackened and mildew rotted. Isolates of Aspergillus, F usarium and known species of yeasts were prepared from the deteriorated discs. The discs containing the diethyl alpha allyl phosphonate fungal inhibitor were not adversely affected.

EXAMPLE XI Algaecidal Properties of Diethyl Alpha Allyl Phosphonate The effectiveness of diethyl alpha allyl phosphonate against various algae species was established in the following manner. Algal cultures representing Scenedesmus, Chlorella, Plectonema, Anacystis, Ankistrodesmus, Anabaena, Synura, Oscillatoria, Coccochloris, Chlamydomonas and Lyngbya were each maintained in Chu No. 10 Broth Medium (Calcium nitrate, 0.040 grams; Potassium phosphate, 0.010 grams; Magnesium sulphate, 0025 grams; Sodium carbonate, 0.020 grams; Sodium silicate, 0.025 grams; Ferric citrate, 0.003 grams; Citric acid, 0.003 grams; and deionized water; 1,000 ml. in the presence of sunlight. Hardened Chu No. 10 agar plates were inoculated with cotton swabs saturated with the respective algae broth cultures. The diethyl alpha allyl phosphonate was tested by impregnating filter paper discs (1.27 cm. in diameter, No. 740-E, Schleicher and Schuell, Keene, New Hampshire) with a solution of the diethyl alpha allyl phosphonate at the concentrations indicated. The saturated filter discs were placed on the surface of the seeded agar plates and the optimum growth temperature of 25 to 27" C. was maintained. The results of these tests are expressed as inhibition zone diameters.

Inhi- Inhi- Inhibition bition bition Zone Zone Zone (cm) (cm) (cm) Algae for for for ug/ l0 ug/ 3 ug/ disc disc disc Scenedesmus basilcnsis l .7 .75 0 Taft EEC 83 Scenedesmux obliquur 2.0h l.0h 0 Taft EEF 92 Scenedesmus uhliquus 4.7 6.4 0 SR] EEC 315 Chlorella vulguris tr 0 0 ATCC No. 9765 Placelrmema nolalum 2.5 L8 1.0 Taft EEC l72 Anacyslis nidulanx tr 0 O Taft EEC I34 Ankixlrodesmu: var. acicularis Taft EEC 28 2.0 1.1 0 Anabaena calsnula 4.7 4.0 2.5 SRl EEC 3l5 Synum sp. 5.0 5.0 0 UI Oscillaloria camen' 1.8 1.0 0 UI Cacwchloris elebans I 7 0 0 SR! Chlamydomnnas radiali 1.8 L0 0 UA Lyngbya .rp. l 9 0 0 Taft EEC I66 CommentszH hazy zone As was the case with the bacteria, it should be recognized that the scope of this invention should not be limited to any particular species. For instance, the algaecidal activity of diethyl alpha allyl phosphonate against Chlorella vulgaris suggests that the compound will also prove to be of value against such other Chlorella species such as Chlorella ellipsoidea, Chlorella pyrenoidosa, Chlorella variegata, et cetera. Similar possibilities exist for species of other genera whose activity was shown to be arrested by diethyl alpha allyl phosphonate. It should be recognized also that other appropriate algae genera may well afford additional opportunities to further define the degree and spectrum of the algaecidal activity disclosed in this invention. Since no firm procedure can be laid down for the sequencing of such evaluations or for a selection among the more than 20,000 known algae species, the diethyl alpha allyl phosphonate compounds of this invention must be considered on the basis of their demonstrated performance in these primary evaluations and then progressively judged in subsequent studies. These evaluations should include but not be limited to the following algae genera.

TASTE AND ODOR CAUSlNG ALGAE GENERA Astcrionclla Peridinium Nitella' Anabaena Mallomonas Dinobryon Microcystis Aphanizomenon Volvox Uroglenopsis Staurastrum Pandorina Hydrodictyon Ceratium Synura Synedra Coelosphaerium CLEAN WATER ALGAE GENERA Rhizocloniurn Merismopedia Meridion Pinnularia Aphanothece Chromulina Cladophora Ulothrix Phacotus Rhodomonas Navicula Staurastrum Surirella Chamaesiphon Lemanea Cyclolella Micrasterias Cncconeis Chrysococcus Calothrix Microcoleus Ankistrodesmus FILTER CLOGGING ALGAE GENERA Anabuena Closterium Spirogyra Chroococcus Tabellaria Trachelomonas Dinobryon Rivularia Aslerionella Cymbella Melosira Palmella Chlorella Cyclotella Diatoma Synedra Navicula Fragilarai Tribonema Oscillatoria POLLUTED WATER ALGAE GENERA Arthrospira Tetraedron Anabaena Mcrismopedia Euglena Phacus Phormidium Spirogyra Gloeogapsa Carteria Chlorococcum Stigeoclonium Lepocinclis Osillatoria Gomphonema Nilzschia Lyngbya Chlamydomonas Chlamydobotrys SURFACE WATER ALGAE GENERA Actinastrum Euastrum Zygnema Nodularia Gonium Stauroneis Coelastrum Desmidium Sphaerocystis Euglena Pediastrum Scenedesmus Micractinium Eudorina Oocystis Mougeotia Gomphnsphaeria RESERVOIR ALGAE GENERA Chara Audouinella Compsopogon Phormidium Tctraspora Bathrachospcrmum Ulothrix Achnanthes Cymbella Cladophora Stigeoclonium Bulbochaete Gomphonema Lyngbya Draparnaldia The scope of this invention should encompass the use of this compound in waters of all types such as lakes, rivers, ponds, streams, reservoirs, swimming pools and oceans as well as recirculating industrial waters. These compounds can be used to prevent the initial occurrence of algae or they can be used on bodies of water with algae already blooming. The diethyl alpha allyl phosphonate compounds of the present invention are also advantageous in that they are degradable with none of the degradation products being toxic to fish and most fish food organisms at algae killing concentrations.

Another important advantage of diethyl alpha allyl phosphonate compounds in their algaecidal applications is that they can be made up in solid or liquid formulations. Examples of solid formulations are dust, wettable powders, granules and pellets. Solid formulations, particularly floating solid formulations, may be preferred in combating algae which grow on surface waters. As a dust, diethyl alpha allyl phosphonate compounds may be dispersed in powdered solid carriers such as talc, soap, soapstone, attapulgus clay, as well as other finely divided solids. When formulated as wettable powders, the active diethyl alpha allyl phosphonate component may be employed in conjunction with inert fillers which may be of the clay type carrier or non-clay type in conjunction with various combinations of wettingv agents and emulsifiers which permit adaptation as a free flowing powder. Each of these carriers may in turn be combined with other carriers which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like, as well as diatomaceous earth, micas or other suitable materials.

When liquid formulations are desirable, liquid extenders or carriers of a non-reactive nature may be utilized. These compositions should contain approximately 0.1- to 20 percent by weight and preferably 0.1 to 3 percent and most preferably 0.5 to 2 percent of the active diethyl alpha allyl phosphonate ingredient. Solvents which may be used in the preparation of such compositions would include alcohols, ketones, glycols, mineral spirits and aromatic solvents such as benzene, xylene, nitrobenzene, dimethylformide. Furthermore, to assist in the rapid and complete dispersion in water systems, these compositions may also contain approximately 5 to 30 percent by weight and preferably 10 to 15 percent by weight of surface-active agents. Suitable surface-active agents include sodium dialkyl sulphates, sodium alkylbenzene sulfonate's, sodium carboxylates and the non-ionic surfactants such as ethoxylated fatty acid alcohols and amines.

Diethyl alpha allyl phosphonate is compatible with a wide variety of other algaecidal, biocidal and fungicidal materials. For example, it may be convenient to combine one or more diethyl alpha allyl phosphonate compositions with one or more of the other biocides, fungicides or algaecides. For example, common fungicides and biocides such as sulphur, inorganic salts such as copper sulphate, activated colloidal silver compounds, copper naphthenate and zinc acetate, as well as substituted hydrocarbons and quartemary ammonium compounds, may be employed.

Considerations concerning concentrations and method of applications may make some methods of application and use of diethyl alpha allyl phosphonate more favored than others. These considerations may include the type of organisms on which the compound is to be administered, the degree of activity, the degree of inhibition toward the algae organism and possible side effects. Also to be considered is the cost of production and the characteristic solubility of diethyl alpha allyl phosphonate in the carrier compounds.

In their algaecidal aspects the applicants have discovered that diethyl alpha allyl phosphonate compounds are active algaecides at relatively low concentrations. For example, it has been discovered that these compounds have imparted algaecidal activity at concentrations as low as 0.1 ppm. The amount of diethyl alpha allyl phosphonate added to the water will, of course, vary depending upon such factors as the type of algae present, the nature of the body of water, i.e. flowing stream versus small lake, et cetera, and the inherent ability of the body of water to support algae growth. This inherent ability in turn depends upon such factors as exposure to sunlight, pH, nutrient capabilities, and the like. In most cases, however, the concentration of diethyl alpha allyl phosphonate required to kill or inhibit growth of algaes will vary from 0.1 to 10 ppm, the preferred amount is in the range of 0.8 to 5 ppm. These compounds can be added to the water according to conventional techniques for algaecide applications. When treating a lake or body of water which is relatively calm, the conventional procedure is to spray an aqueous solution of the algaecide over the surface of the water. The diethyl alpha allyl phosphonate generally will be predissolved in the types of solvents previously mentioned. In the case of moving water, such as in a water treatment plant or industrial facility, the algaecide can be added to the water in small amounts at periodic intervals. For economic reasons, volume usages such as in lakes, streams, reservoirs, as distinguished from specialized uses such as in aquatic gardens, and industrial applications, the concentrations of the diethyl alpha allyl phosphonate algaecides probably will not be more than 0.8 to 5 ppm of the water containing the algae.

Having thus disclosed our invention, we claim:

1. A method of killing or inhibiting the growth of microorganisms selected from the group consisting of fungi, Gram positive bacteria and Gram negative bacteria which comprises contacting said microorganisms with diethyl alpha allyl phosphonate in an amount effective to kill or inhibit the growth of said microorganisms.

2. The method of claim 1 wherein the Gram positive bacteria are selected from the group consisting of Staphylococci, Corynebacter, Listeria, Micrococci, M ycobaterium Di plococci S treptococci and Xanthomonas.

3. The method of claim 2 wherein the Staphylococci is Staphylococcus aureus.

4. The method of claim 2 wherein the Corynebacter are selected from the group consisting of Corynebacterium diphtheriae and Corynebacterium haemolyticum.

5. The method of claim 2 wherein the Listeria is Listeria monocytogenes.

6. The method of claim 2 wherein the Micrococci are selected from the group consisting of Micrococcus tetragena, Micrococcus melitensis and Micrococcus lysodeikticus.

7. The method of claim 2 wherein the Mycobacterium are selected from the group consisting of M ycohacterium avium, Mycobacterium bovis, M ycohacterium phlei, M ycobacterium flrrtuilum, and M ycohacteri um bu t yricum 8. The method of claim 2 wherein the Diplococci are selected from the group consisting of Diplococcus intracellularis and Diplococcus pneumoniae.

9. The method of claim 2 wherein the Streptococci are selected from the group consisting of Streptococcus hemolytic group A, and Streptococcus hemolytic group B.

10. The method of claim 2 wherein the Xanthomonas are selected from the group consisting of Xanthomonas vesicatoria, and Xanthomonas phaseoli.

11. The method of claim 1 wherein the Gram negative bacteria are selected from the group consisting of Escherichia, Shigella, Salmonella, Vibrio, Neisseria, Haemophilus, Bacteriodes, Brucella, Proteus, Pseudomonas and Erwinia.

12. The method of claim 11 wherein the Escherichia is Escherichia coli.

13. The method of claim 11 wherein the Shigella are selected from the group consisting of Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnet.

14. The method of claim 11 wherein the Salmonella are selected from the group consisting of Salmonella derby, Salmonella enteritis, Salmonella gallinarium, Salmonella panama, Salmonella paratyphi, Salmonella pullorum and Salmonella typhosa.

15. The method of claim 11 wherein the Vibrio are selected from the group consisting of Vibrio cholerae and Vibrio fetus.

16. The method of claim 11 wherein the Neisseria are selected from the group consisting of Neisseria gonorrhoeae, Neisseria intracellularis and Neisseria meningitides.

17. The method of claim 11 wherein the Hemophilus are selected from the group consisting of Hemophilus hemolyticus, Hemophilus influenzae, Hemophilus parainfluenzae, Hemophilus suis and Hemophilus vaginalis.

18. The method of claim 11 wherein the Bacteroides are selected from the group consisting of Bacteroides nigrescens, Bacleroides pneumosintes and Bacteroides serpens.

19. The method of claim 11 wherein the Brucella are selected from the group consisting of Brucella abortus, Brucella melitensis and Brucella suis.

20. The method of claim 11 wherein the Proteus are selected from the group consisting of Proteus vulgaris, Proteus morganii and Proteus mirabilis.

21. The method of claim 11 wherein the Pseudomonas are selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas maltophilia, Pseudomonas K997 and Pseudomonas K955.

22. The method of claim 11 wherein the Erwinia is Erwinia carotovora.

23. The method of claim 1 wherein the microorganisms are present on animals or plants and are contacted with an effective amount of diethyl alpha allyl phosphonate together with an acceptable carrier.

24. The method of claim 1 wherein the fungi are selected from the group consisting of Fusarium, Penicillium, Aspergillus, Alternaria, Rhizopus, Candida, Rhodotorula, Monascus, Scopulan'opsis, Cephalosporium, Phoma, Paecilomyces, Nigrospora, Absidia, Thamnidium, Phycomyces, Beauvaria, Phialophora, Cladosporium, Torula, Monosporum, Geotrichum, Verticillium, Trichophyton, Cercospora, Pythium, Helminlhosporium and Micro-sporum.

25. The method of claim 24 wherein the Fusarium is selected from the group consisting of F usarium roseum, Fusarium oxysporum and F usarium oxysporum cubense.

26. The method of claim 24 wherein the Penicillium is selected from the group consisting of Penicillium rubrum and Penieillium notatum. i

27. The method of claim'24 wherein the Aspergillus is selected from the group consisting of Aspergillus, niger, Aspergillus sydowi, Aspergillus nidulons, Aspergillus flavas, Aspergillus amstelodami, and Aspergillus fumiga- 28. The method of claim 24 wherein the Alternaria is Alternaria solani.

29. The method of claim 24 wherein the Rhizopus is Rhizopus stolonzfer.

30. The method of claim 24 wherein the Candida is selected from the group consisting of Candida albicans, Candida krusei, Candida quilliermondii, Candida tropicalis, Candida pseudatrapicalis, Candida pulcher-rima, Candida inlermedia and Candida zeylanoides.

31. The method of claim 24 wherein the Rhmloturula is Rhodotorula Sp. Duke.

32. The method of claim 24 wherein the Monascus is Monascus purpurea.

33. The method of claim 24 wherein the Scopulaniopsis is Scapulariopsis sp. U.

34. The method of claim 24 wherein the Cephal osporium is selected from the group consisting of 25 the Absidia spinosa.

39. The method of claim 24 wherein the Mucor is Mucor racemosus.

40. The method of claim 24 wherein the Thamnidium is Thamnidium elegans.

41. The method of claim 24 wherein the Phycomyces is Phycomyces nitens.

42. The method of claim 24 wherein the Beauvaria is selected from the group consisting of Beauvaria bassiana and Beauvaria tenella.

43. The method of claim 24 wherein the Philophora is Phialophora verrucosa.

44. The method of claim 24 wherein Cladosporium is Cladosporium trichoides.

45. The method of claim 24 wherein the Torula is Torula bergerL 46:. The method of claim 24 wherein Monosporum is Monosphurum apiospermum.

47. The method of claim 24 wherein the Verticillium is Verticillium albo-atrum.

48. The method of claim 24 wherein the Trichophymn are selected from the group consisting of Trichaphyton mentagrophytes and Trichophyton tonsuruns.

49. The method of claim 24 wherein the Cercaspora is Cercospora beticola.

50. The method of claim 24 wherein the Pythium is Iylhium arrlu'nomam's.

the

the

5 l The method of claim 24 wherein the Helminthosporium is Helminthosporium oryzae.

52. The method of claim 24 wherein the Microsporum is Micrasporum gypseum. 

2. The method of claim 1 wherein the Gram positive bacteria are selected from the group consisting of Staphylococci, Corynebacter, Listeria, Micrococci, Mycobaterium, Diplococci, Streptococci and Xanthomonas.
 3. The method of claim 2 wherein the Staphylococci is Staphylococcus aureus.
 4. The method of claim 2 wherein the Corynebacter are selected from the group consisting of Corynebacterium diphtheriae and Corynebacterium haemolyticum.
 5. The method of claim 2 wherein the Listeria is Listeria monocytogenes.
 6. The method of claim 2 wherein the Micrococci are selected from the group consisting of Micrococcus tetragena, Micrococcus melitensis and Micrococcus lysodeikticus.
 7. The method of claim 2 wherein the Mycobacterium are selected from the group consisting of Mycobacterium avium, Mycobacterium bovis, Mycobacterium phlei, Mycobacterium fortuitum, and Mycobacterium butyricum.
 8. The method of claim 2 wherein the Diplococci are selected from the group consisting of Diplococcus intracellularis and Diplococcus pneumoniae.
 9. The method of claim 2 wherein the Streptococci are selected from the group consisting of Streptococcus hemolytic group A, and Streptococcus hemolytic group B.
 10. The method of claim 2 wherein the Xanthomonas are selected from the group consisting of Xanthomonas vesicatoria, and Xanthomonas phaseoli.
 11. The method of claim 1 wherein the Gram negative bacteria are selected from the group consisting of Escherichia, Shigella, Salmonella, Vibrio, Neisseria, Haemophilus, Bacteriodes, Brucella, Proteus, Pseudomonas and Erwinia.
 12. The method of claim 11 wherein the Escherichia is Escherichia coli.
 13. The method of claim 11 wherein the Shigella are selected from the group consisting of Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei.
 14. The method of claim 11 wherein the Salmonella are selected from the group consisting of Salmonella derby, Salmonella enteritis, Salmonella gallinarium, Salmonella panama, Salmonella paratyphi, Salmonella pullorum and Salmonella typhosa.
 15. The method of claim 11 wherein the Vibrio are selected from the group consisting of Vibrio cholerae and Vibrio fetus.
 16. The method of claim 11 wherein the Neisseria are selected from the group consisting of Neisseria gonorrhoeae, NeisSeria intracellularis and Neisseria meningitides.
 17. The method of claim 11 wherein the Hemophilus are selected from the group consisting of Hemophilus hemolyticus, Hemophilus influenzae, Hemophilus parainfluenzae, Hemophilus suis and Hemophilus vaginalis.
 18. The method of claim 11 wherein the Bacteroides are selected from the group consisting of Bacteroides nigrescens, Bacteroides pneumosintes and Bacteroides serpens.
 19. The method of claim 11 wherein the Brucella are selected from the group consisting of Brucella abortus, Brucella melitensis and Brucella suis.
 20. The method of claim 11 wherein the Proteus are selected from the group consisting of Proteus vulgaris, Proteus morganii and Proteus mirabilis.
 21. The method of claim 11 wherein the Pseudomonas are selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas maltophilia, Pseudomonas K997 and Pseudomonas K955.
 22. The method of claim 11 wherein the Erwinia is Erwinia carotovora.
 23. The method of claim 1 wherein the microorganisms are present on animals or plants and are contacted with an effective amount of diethyl alpha allyl phosphonate together with an acceptable carrier.
 24. The method of claim 1 wherein the fungi are selected from the group consisting of Fusarium, Penicillium, Aspergillus, Alternaria, Rhizopus, Candida, Rhodotorula, Monascus, Scopulariopsis, Cephalosporium, Phoma, Paecilomyces, Nigrospora, Absidia, Thamnidium, Phycomyces, Beauvaria, Phialophora, Cladosporium, Torula, Monosporum, Geotrichum, Verticillium, Trichophyton, Cercospora, Pythium, Helminthosporium and Micro-sporum.
 25. The method of claim 24 wherein the Fusarium is selected from the group consisting of Fusarium roseum, Fusarium oxysporum and Fusarium oxysporum cubense.
 26. The method of claim 24 wherein the Penicillium is selected from the group consisting of Penicillium rubrum and Penicillium notatum.
 27. The method of claim 24 wherein the Aspergillus is selected from the group consisting of Aspergillus, niger, Aspergillus sydowi, Aspergillus nidulons, Aspergillus flavus, Aspergillus amstelodami, and Aspergillus fumigatus.
 28. The method of claim 24 wherein the Alternaria is Alternaria solani.
 29. The method of claim 24 wherein the Rhizopus is Rhizopus stolonifer.
 30. The method of claim 24 wherein the Candida is selected from the group consisting of Candida albicans, Candida krusei, Candida quilliermondii, Candida tropicalis, Candida pseudotropicalis, Candida pulcher-rima, Candida intermedia and Candida zeylanoides.
 31. The method of claim 24 wherein the Rhodotorula is Rhodotorula sp. Duke.
 32. The method of claim 24 wherein the Monascus is Monascus purpurea.
 33. The method of claim 24 wherein the Scopulariopsis is Scopulariopsis sp. OU.
 34. The method of claim 24 wherein the Cephalosporium is selected from the group consisting of Cephalosporium acremonium and Cephalosporium sp. OU.
 35. The method of claim 24 wherein the Phoma is Phoma pigmentovora.
 36. The method of claim 24 wherein the Paecilomyces is Paecilomyces varioti.
 37. The method of claim 24 wherein the Nigrosphora is Nigrosphora sphaerica.
 38. The method of claim 24 wherein the Absidia is Absidia spinosa.
 39. The method of claim 24 wherein the Mucor is Mucor racemosus.
 40. The method of claim 24 wherein the Thamnidium is Thamnidium elegans.
 41. The method of claim 24 wherein the Phycomyces is Phycomyces nitens.
 42. The method of claim 24 wherein the Beauvaria is selected From the group consisting of Beauvaria bassiana and Beauvaria tenella.
 43. The method of claim 24 wherein the Philophora is Phialophora verrucosa.
 44. The method of claim 24 wherein the Cladosporium is Cladosporium trichoides.
 45. The method of claim 24 wherein the Torula is Torula bergeri.
 46. The method of claim 24 wherein the Monosporum is Monosphorum apiospermum.
 47. The method of claim 24 wherein the Verticillium is Verticillium albo-atrum.
 48. The method of claim 24 wherein the Trichophyton are selected from the group consisting of Trichophyton mentagrophytes and Trichophyton tonsurans.
 49. The method of claim 24 wherein the Cercospora is Cercospora beticola.
 50. The method of claim 24 wherein the Pythium is Pythium arrhenomanes.
 51. The method of claim 24 wherein the Helminthosporium is Helminthosporium oryzae.
 52. The method of claim 24 wherein the Microsporum is Microsporum gypseum. 